It is well known that an inflamed area is hyperalgesic. However, the mechanism underlying sensitization is not understood. Our overall goal is to understand how the process of sensitization occurs, how it can be interrupted, and to define the chemical identity of the neurons that become sensitized. The mechanism of sensitization is likely to involve an interplay between primary afferent neurons and substances generated by the immune system since sensitization is generally associated with tissue damage. In these exploratory studies we will focus on whether eicosinoids and cytokines act directly on primary afferent neurons to enhance the response of these neurons to the algogens bradykinin and serotonin. Since different populations of primary afferent neurons may respond differently to sensitizing substances, we will also define some of the neurochemical characteristics of the sensory neurons that are assayed for their responsiveness. The most relevant portion of the primary afferent neuron for studies of sensitization is its peripheral process, and specifically, the peripheral terminal. The technical evolution of activity dependent fluorescent dyes, and microfluorimetric detection systems for these dyes, makes it possible to study biochemical changes in individual processes of primary afferent neurons. We propose to use Indo-1 to measure changes in intracellular Ca2+ ([Ca2+]i) as a bioassay. Since depolarization of primary afferent neurons opens voltage-sensitive calcium channels in the neuronal membrane, changes in [Ca2+]i will provide an indirect measure of neuronal depolarization. Our experiments will address the following questions: 1. Do substances associated with inflammation increase [Ca2+]i in primary afferent neurons? The activity of two prostaglandins (PGE2 and PGI2) and two cytokines (interleukin 1-beta and tumor necrosis factor-alpha) will be explored. 2. Do prostaglandins and cytokines potentiate the action of known algogens in causing changes in [Ca2+]i? Bradykinin and serotonin depolarize sensory neurons and cause overt pain. These algogens also increase [Ca2+]i. Since prostaglandins and cytokines may not have a direct effect on [Ca2,]i, we will determine whether these substances potentiate the [Ca2+]i that occurs in response to bradykinin and serotonin. 3. What populations of primary afferent neurons, based on their peptide content, exhibit sensitization? We will determine whether the neuronal processes that were assayed contained calcitonin gene-related peptide and/or substance P immunoreactivity. The occurrence of RT97 immunoreactivity, which is a predictor of axon myelination, will also be determined. The above studies will initially be conducted in primary cultures of neonatal rat dorsal root ganglion neurons. We will also explore whether these studies can be conducted in vitro on peripheral processes of sensory neurons in tooth pulp obtained from adult rats.